JP2002504335A - Pollen-specific promoter - Google Patents

Abstract

  (57) [Summary] A promoter sequence of the ZmC5 gene in maize, or a variant or fragment thereof that acts as a promoter in pollen. The DNA sequence of this promoter is shown in FIG. Also claimed are expression cassettes and expression systems, transformation methods, and transformed plants containing the promoter sequences of the present invention. These nucleic acids can be used, inter alia, for the production of male-sterile plants and / or hybrids, and for the transformation of pollen.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to pollen-specific promoter sequences, constructs containing the promoter and transgenic plant cells and plants, and methods for transforming pollen using this promoter and controlling fertility in plants. .

[0002] To introduce desired genetic traits from two plants into a single plant, for example,
Various cross breeding or hybrid cross breeding represent a traditional approach. To ensure a consistent hybrid, it is necessary to ensure that self-pollination of the parent plant does not occur.

[0003] This can be achieved by ensuring that one parental line is male-sterile. Various techniques for producing male sterility are known and have been proposed in the art. One method involves the removal of either anthers or tassels of the female parent plant, either manually or mechanically. The plant can then be pollinated only by pollen from the sire, so that its progeny are hybrids. But,
Such a process is labor intensive and totally unreliable. Because some female plants may miss the detasseling process, or in some cases they will develop secondary tassels after the detasseling is complete. In addition, in this system, the male parent is self-pollinating, and therefore it is necessary to physically separate the male and female parents to allow for easy harvesting of the hybrid seed. This prevents proper planting of corn.
Because pollen is light and produced in large quantities. However, this approach is not applicable to species with heavier pollen, and in species where male and female flower organs are contained within one flower (eg, wheat, rice).

[0004] Chemical methods for preventing pollen production are also known. US Patent No. 4,801,3
No. 26 describes chemicals that can be applied to plants or soil to prevent pollen production. However, again, such techniques are labor intensive and are not totally reliable. It is imperative that sufficient chemicals be applied at appropriate time intervals to ensure that pollination does not occur, with attendant loads on the environment. In addition, these chemicals are very expensive.

[0005] Genetic engineering has been provided as an alternative whereby male sterility can be produced, maintained, and / or subsequently restored in a plant.

[0006] Systems have been described that include an inducible promoter that allows pollination to be switched on or off based on the application or availability of an external compound. For example, WO 90/08830 describes the induction of male sterility in plants by a cascade of gene sequences expressing a protein that disrupts pollen biosynthesis.
WO93 / 18171 discloses a GST promoter that inducibly expresses chalcone synthase (chs) and restores fertility to male sterile plants that are rendered sterile by "knocking out" the endogenous chs gene. Describe use.

[0007] A different approach has been described in WO 93/25695 (PGS). This is based on the use of a tapetum-specific promoter that expresses the pollen lethal gene, barnase, in tapetal cells. These cells are
Destroy pollen production as it is important for pollen development. Balstar, a healing gene
barstar) can be used to restore fertility (Mariani et al., Nature, 357: 384-387).

[0008] There is an advantage of expressing a gene that directly affects pollen production in pollen in a controllable manner. Suitably, fertility should be restored if desired.

A component of the system for genetically manipulating male sterility is the effectiveness of a promoter to specifically drive expression in either pollen or tissue on which pollen production or development is dependent.

[0010] Many of the characterized genes that are specifically or highly expressed in pollen and germinating pollen encode proteins that play a role in cell wall metabolism, for example, as follows: Which have homology to genes encoding enzymes involved in pectin degradation: polygalacturonase (SM Brown et al., Plant Cell (1990) 2: 263-274;
SJ Tebbutt et al., Plant Mol. Biol. (1994) 25: 2.
83-299), pectate lyase (HJ Rogers et al., Plant Mo.
l. Biol 20: 493-502 (1992), RA Wing et al., Plan.
t Mol. Biol 14: 17-28 (1989)), and pectin methylesterase (PME) (JH Mu et al., Plant MoI. Biol 25: 539-544 (1994)).

[0011] Other genes that are highly expressed in pollen include genes that encode: cytoskeletal proteins (I. Lopez et al., Proc. Natl,
Acad Sci USA 93: 7415-7420 (1996), HJ Log.
ers et al., Plant J .; 4: 875-882 (1993) and CJ Sta.
iger et al., Plant J .; 4: 631-641 (1993)), a putative ascorbate oxidase, a Kunitz protein inhibitor, and many other proteins whose function cannot be inferred by homology to known genes.
The temporal expression of such genes has been studied, and many have been found to be expressed late in microsporogenesis and to be maximal in mature microsporocytes. In some cases, continuous expression in pollen tubes has also been demonstrated (AK Kononowicz et al., Plant Cell 4: 513-524 (1992)). These genes are called "late genes." Most of the expression at this stage is from vegetative cells rather than germ cells, and most of these "late" genes appear to be transcribed from vegetative nuclei: It has only been demonstrated for the "late" gene (D. Twel
1, Plant J2: 887-892 (1992)). Distinct classes of genes expressed in anthers have different expression programs, are first detectable shortly after the tetrad stage, and are found to decay appropriately in expression before pollen maturation ing. The major role of these "early" genes may be during microspore differentiation and development, rather than pollen tube growth. further,
US Pat. No. 5,086,169 (Mascarenhas) describes the isolation of a first pollen-specific promoter from corn.

[0012] Applicants have isolated additional promoters that are specifically expressed only in pollen tissue. This promoter is derived from the "late" pollen expression gene, ZmC5, isolated from maize.

Thus, according to a first aspect of the present invention there is provided a recombinant nucleic acid comprising a promoter sequence of the ZmC5 gene in maize, or a variant or fragment thereof, which acts as a promoter in pollen.

As used herein, the term “fragment” includes one or more regions of a nucleotide sequence that retain promoter activity. Here, a fragment comprises one or more regions, which may be directly ligated together or may be separated by additional bases.

An expression “variant”, in reference to the present invention, means any substitution, alteration, modification, substitution, deletion or loading of one or more nucleotides from or in a nucleic acid sequence and is obtained The sequence shown indicates the expression of the pollen promoter. The term also includes sequences that are capable of substantially hybridizing to the nucleic acid sequence.

As used herein, the expression “ZmC5 gene” refers to a corn gene that encodes a 563 amino acid sequence described herein. The cDNA sequence encoding this sequence is defined in EMBL Y13285.

[0017] The promoter sequence of the present invention comprises a National Collection of
NCIMB at Industrial and Marine Bacteria
No. 40915 is included in the clone deposited on Jan. 26, 1998. This is a SalI fragment derived from that described herein below. This promoter region is within a region consisting of about 2 kb of sequence upstream of the transcription start site of the corn ZmC5 gene, shown in FIG. 1 herein below.

In accordance with a preferred embodiment of the present invention, at least a portion of the DNA sequence shown in FIG. 5 or at least a portion of a sequence encoding a promoter having activity substantially similar to the promoter encoded in FIG. A recombinant nucleic acid sequence comprising a promoter sequence comprising the same, or a variant or fragment thereof is provided.

The term “substantially similar activity” includes a DNA sequence that is complementary to and hybridizes to the DNA of the present invention and encodes a promoter that acts in pollen. Preferably, such hybridization occurs under conditions of low and high or stringency in between. In general terms, low stringency conditions can be defined as 3 × SCC at an ambient temperature of about 60 ° C. to about 65 ° C., and high stringency conditions can be defined as 0.1 × SS at about 65 ° C.
C. SSC is a buffer of 0.15 M NaCl, 0.015 M trisodium citrate. 3 × SSC is three times the intensity of SSC or the like.

The pollen-specific promoter of the present invention drives male genes that can interfere with pollen production or viability, to manipulate male sterility, or to specifically express a gene of interest in pollen grains Can be used to

According to a second aspect of the present invention, the promoter sequence may form part of an expression cassette in combination with a gene whose expression in pollen, especially in late pollen production, may be desired. These include pollen or genes that have an effect on pollen production. Such genes may be genes involved in controlling male fertility, genes encoding insecticidal toxins, which are then targeted to insect species that feed on pollen, or enhance or modify the nutritional value of pollen Gene. In addition, the promoter sequence can be used to drive expression of a selectable marker for use in pollen transformation. Examples of suitable selectable marker genes include antibiotic resistance genes, such as the kanamycin resistance gene, the hygromycin resistance gene and the PAT resistance gene, which are used to transform stable transformants into species (eg, corn, rice, Wheat).

The terms “expression cassette” (which includes “DNA constructs,” “hybrids” and “
Synonymous with terms such as "conjugate") include effector genes that are directly or indirectly connected to a regulated promoter to form a cassette. An example of an indirect connection is the provision of a suitable spacer group, such as an intron sequence intermediate, a promoter and a target gene. The DNA sequences can also be on different vectors and therefore need not be located on the same vector. The same applies in the context of the present invention for the term "fused", which includes direct or indirect connections. Such constructs also include the appropriate plasmids and phage to transform the cells of interest.

According to a preferred embodiment, the expression cassette of the invention comprises a promoter sequence as described above, which comprises genes that are detrimental to pollen development (eg barnase, adenine nucleotide transporter, mutant tubulin, T-urf ( WO 97/04116
Or a gene encoding trehalose phosphate phosphatase (TPP)).

For example, WO 93/25695 describes the use of the gene barnase, which encodes a cytotoxic protein (under the control of a tapetum-specific promoter). The expression of barnase in tapetum cells destroys these cells and leads to a disruption of pollen production.

A ribozyme is an RNA molecule that can catalyze a nucleotide strand cleavage reaction. These can catalyze the reaction in trans and can be targeted to different sequences. Therefore, they are potential alternatives to antisense as a means of regulating gene expression. Hasselhof and Garlach ((1988) Nat.
ure 334: 585-591) or Wegener et al. ((1994) Mol Gen Genet 245: 465-470) have demonstrated the generation of transdominant mutations by expression of ribozyme genes in plants. If necessary,
The pollen-specific promoter of the present invention can be used to control the expression of a ribozyme that is specifically expressed in pollen.

Baulcombe (1997) describes a replicable viral RNA vector (A
A method of gene silencing in transgenic plants is described by the use of Mmplicons ^™, which may also be useful as a means of knocking out endogenous gene expression. This method has the advantage that it produces a dominant mutation, ie, can be stored in a heterozygous state, and knocks out all copies of the targeted gene, and can also knock out isoforms. This is a clear advantage in wheat, which is hexaploid.
Fertility can then be restored by using an inducible promoter to drive expression of a functional copy of the knockout gene. By including a pollen-specific promoter as an element of the Amplicon ^™ vector, gene expression then occurs specifically in the pollen.

[0027] The use of a cytotoxic or disruptive gene as a means of disrupting pollen production requires the expression of a restored gene to restore fertility. Suitably, the construct is further a nucleotide sequence capable of overcoming the effect of the deleterious gene (eg, a barrestase in the case of barnase or a recovery gene such as TPS in the case of TPP), or is sense or antisense to the deleterious gene It contains a cassette containing the sequence encoding the construct.

An alternative means of controlling the expression of a deleterious gene is to use an operator sequence. The operator sequence (eg, lac, tet, 434, etc.)
No. 08830, can be inserted into the promoter region. Then
Repressor molecules can bind to these operator sequences and prevent transcription of downstream genes, such as genes that are detrimental to pollen development (Wild et al. (19)
92) EMBO J. 11, 1251). In addition, the operator sequence can be manipulated with enhanced binding capacity, such as a Lac IΔHis mutant, as described in (Lehming et al. (1987) EMBO J. 6, 3145-3153). is there. This has an amino acid change at position 17 from tyrosine to histidine, thus giving tight control of expression. When using a combination with inducible expression of a repressor, this then allows the expression of the inactivated gene to be blocked.

In this method, the expression cassette can be incorporated into an expression system that can be used to control plant fertility as described above.

The term “expression system” means that the system defined above can be expressed in a suitable organ, tissue, cell, or medium. The system may include one or more expression cassettes, and may also include additional components to ensure that expression of the target gene is increased by use of a regulated promoter.

According to a third aspect of the present invention there is provided an expression system comprising: (a) a first promoter sequence which is specifically expressed in pollen; (b) when expressed, A first gene that disrupts pollen biosynthesis under the control of a pollen-specific promoter; (c) a second promoter sequence responsive to the presence or absence of an exogenous chemical inducer; and (d) An element capable of inhibiting the expression of the first gene, or the second
A second gene, operably linked to the promoter sequence of E. coli and encoding any of the elements capable of inhibiting a protein encoded by the first gene under the control thereof.

The above elements (a) and (b) and (c) and (d) can be provided by one or two individual vectors, but are preferably included in the same vector to allow simultaneous separation to be certain. These can be used to transform or co-transform plant cells, allowing appropriate interactions between the elements to take place.

[0033] The second promoter sequence and the second gene provide a chemical "switch" for the first gene. Here, the second promoter sequence is responsive to the presence of the exogenous chemical inducer, and the application of the chemical inducer to the pollen or plant has the effect of switching on the second gene, whereby Offset the effect of one gene.
The absence of a chemical inducer has a similar effect, wherein the second promoter sequence is active only in the absence of the chemical inducer.

Elements (c) and (d) may be a nucleotide sequence capable of overcoming the effects of the deleterious genes described above (eg, a recovery gene such as Barstar in the case of barnase or TPS in the case of TPP), or a deleterious gene or Suitable in the form of an expression cassette comprising a sequence encoding a construct that is sense or antisense to the gene encoding the repressor molecule in the case of an operator sequence used operably interconnected with an inducible promoter.

The expression system of the present invention may further comprise a selectable marker such as a herbicide resistance gene or an antibiotic resistance gene, and the stable transformant may be dependent on the species (eg, corn, rice, wheat). Allow to be identified. The presence of the herbicide resistance gene also allows for the selection of male sterile progeny in segregating populations.

Transformation of a plant using such an expression system results in the production of a male sterile plant,
And the method of producing such a plant forms the fourth aspect of the present invention.

The expression system according to this embodiment of the invention, wherein the gene is detrimental to viable pollen production, is useful for hybrid production, but the male sterile line is homozygous. It is particularly useful if it can be made. If a "late" promoter (eg, the ZmC5 promoter described above) is used, the primary transformants and heterozygotes will have one sterility since this gene product is expressed late in pollen development.
: 1 (ie, 50% of the pollen is fertile, which can result in self-pollination leading to non-hybrid seed).

To obtain a homozygous sterile plant, the inducible promoter uses an appropriate chemical (eg, ethanol for the AlcA / R switch, or a safener for the GST switch). Must be switched to drive expression of the recovery gene. This in turn inactivates the deleterious gene, thus allowing self-pollination to occur according to the following model.

[0039]

[Table 1] All pollens from homozygous progeny (MSMSRcsRcs) are sterile.
50% of the pollen from heterozygous progeny (MSRcs--) is sterile.
All of the pollen from the invalid (------) progeny is fertile. Staining of pollen from these lines using live staining, such as DAPI, allows the identification of plants that produce 100% sterile pollen. Alternatively, induction and selfing can be repeated in this segregating population and progeny can be analyzed for sterility segregation.
Clearly, all progeny that are derived from self-pollination of homozygous lines are homozygous in themselves and are male-sterile. That is, they all produce pollen that is 100% sterile, while progeny resulting from self-pollination of heterozygous lines continue to segregate for sterility. Alternatively, if the gene that causes male sterility is linked to a gene that confers herbicide tolerance, the progeny can be sprayed with herbicide at an early seedling stage and herbicide tolerance can be separated from the sterility gene. Is done. In this manner, male sterile parents can be identified and selected for hybrid production.

[0040] Once identified, this homozygous sterile line can be used to produce an F1 hybrid by cross-pollination by an unmodified inbred male parent line. See below.

[0041]

[Table 2] Thus, all F1 seeds are hybrid and heterozygous for sterility, which means that 50% of the pollen from each plant is fertile. In crop species such as corn, this is an abundant viable pollen that ensures complete pollination across the field due to the vertical volume of pollen produced by each tassel. Also in wheat, self-pollination occurs while the flowers are still closed, so this should be sufficient pollen to ensure that there is no loss in yield, thus reducing losses due to wind, etc. I do. In the species from which the vegetative part of the plant is harvested, then, reduced pollen viability is not a factor to be considered.

There is also an additional advantage for hybrid seed producers in that farmers should retain F2 seed for later planting. Farmers suffer loss of yield as a result of loss of heterosis. See below.

[0043]

[Table 3] These methods may allow for reversal of sterility, for example, in hybrid production by activation using an inducible promoter.

According to a fifth aspect of the present invention there is provided a method of controlling plant fertility, comprising the steps of transforming said plant with an expression system as described above, and Activating the inducible promoter, if it is to be restored, is included.

Suitable inducible promoters include those that are controlled by the application of an external chemical stimulus (eg, the application of a herbicide safener. Examples of inducible promoters include, for example, two-component systems such as: The alcA / alcR gene switch promoter system described in our International Publication No. WO93 / 21334, the ecdysone switch system described in our International Publication No. WO96 / 37609, or an international patent. Application No. WO90 / 08826
And the GST promoter described in WO 93/031294, the teachings of which are incorporated herein by reference. Such a promoter system is referred to herein as a "switch promoter". The switch chemistry used in combination with the switch promoter is an agriculturally acceptable chemical that makes this system particularly useful in the methods of the present invention.

When the pollen-specific promoter of the present invention is used to obtain male sterility,
Complete restoration of fertility cannot be achieved by this method. Because pollen is haploid. This is 50% of the pollen produced after activation of the recovery gene
Only means fertile. This can be counted by the fact that expression using the promoters of the invention is highly tissue specific.

This property makes the use of the promoter of the invention particularly useful in some very special applications. For example, in some cases, pollen transformation is required. In this case, the use of a pollen-specific promoter may be highly desirable. An example of such an application is MAGE LITER (Male Embryonic Line Transformation).
And Stagger et al. (Plant Cell Report)
s 14 (1995) 273-278). In this method, pollen is transformed by microinjection bombardment. Pollen-specific promoters are used, for example, to drive a selectable marker gene. The fact that the promoter is pollen-specific confers several advantages. First,
In addition, this marker is expressed only in pollen and not in the rest of the plant, so that the remaining plant tissue does not contain the unwanted marker. In addition, having a selectable marker only in pollen allows the possibility of re-transforming by the usual methods and not having to have a different selectable marker
(Ie, allow for easier gene stacking). The reason why pollen transformation is important is that pollen can be subjected to the development of a sporophyte form (ie, yielding haploid and haploid doubled plants). This means that homozygosity is achieved in one step. Alternatively, the transformed pollen re-pollinates the wild-type plant in a faster process than the traditional transformation pathway,
Thus, it can be used to obtain seeds carrying the introduced transgene.

The expression system of the present invention can be introduced into plants or plant cells by any of the available methods, including Agroba containing recombinant Ti plasmid.
infection with C. tumefaciens, electroporation, microinjection of plant cells and protoplasts, microinjection bombardment, bacterial bombardment, certain “fibre” or “whisker” methods, and pollen tube transformation. These are dependent on the particular plant species being transformed. The transformed cells can then, where appropriate, be regenerated into whole plants, where the new nuclear material is stably integrated into the genome. Both transformed monocots and dicots can be obtained in this way. Reference may be made to the literature for all details of known methods. Such a method forms a further aspect of the present invention.

The method of the present invention is useful for the production of a wide range of hybrid plants (eg, wheat, rice, corn, cotton, sunflower, sugar beet, and lettuce, rapeseed and tomato).

Plant cells containing a plant gene expression system as described above, together with plants containing these cells, form a further aspect of the present invention.

According to a ninth aspect of the present invention, a replicable viral DNA vector (Ampl
icon The ^TM) is provided which comprises a recombinant nucleic acid as defined above.

According to a tenth aspect of the present invention, there is provided a method for transforming pollen cells using the above expression system.

The present invention will now be particularly described, by way of example only, with reference to the accompanying drawings.

Example 1 (ZmC5 cDNA and Genomic Clones) Maize (inbred A188) pollen and budding pollen (HJ Rogers et al., Plant J4 (1993) 875-882) cDNA library was used for pollen and shoot poly. (A) -Screened according to the following standard procedure using a cDNA probe generated against RNA (FM Ausubel et al., Current protocols in Molecular Biology).
, Wiley, New York (1990)). All clones showing hybridization to radiolabeled pollen cDNA but not to radiolabeled shoot DNA (total 1,101) were picked. Random colonies were picked and sequenced at the 5 'end, and the sequences were compared to current databases. One clone with an 800 bp insert showed significant sequence identity with a known pectin methylesterase. Pollen c
The DNA library is rescreened using this cDNA insert,
A full length ZmC5 clone (ZmC5c) was identified and sequenced.

A maize (inbred B73) genomic library (8 × 10 ⁶ plaques) was screened using a PCR fragment corresponding to the 270 bp 5 ′ of cDNA clone ZmC5c labeled by random priming (Au
sbel et al., supra). One positive clone, ZmC, 5 g, was plaque purified. Comparison of the sequence of the 2.5 kb SalI fragment of ZmC5g subcloned into pUC19 with ZmC5c (deposited as NCIMB 40915) indicated that the two sequences overlapped and that they were identical. This indicates that this clone displays the same gene. A representation of the overlap between the 2.5 kb fragment of the genomic clone with this cDNA is shown in FIG.

The start of transcription is determined by nucleotides 1-21 (5 ′) of the coding sequence (data not shown).
-ACCTAGGAGAGCCTTTGCCAT-3 ') and 56-82 (5
'-AGCGGGGTGACGGTGGCGACCACACCGA-3') and mapped using two oligonucleotide primers. This product clearly positions the transcription start site on the A nucleotide only 15 bases upstream of the putative ATG (FIG. 1). Other pollen-specific genes have been found to have long 5 'untranslated sequences (SJ Tebbutt et al., Plant Mol. Biol.
1994) 25: 283-299). Thus, this region of ZmC5g appears to be very short. The calculated free energy for this short 5 ′ UTL is 0.9 kJ / mol (M. Zuker, Meths Enzymol. (1989) 180: 262-288), so that no stable secondary structure can be formed. To

A 1692 bp open reading frame was identified and the putative ATG start codon (FIG. 1) is in good agreement with the consensus sequence (HA Lutcke et al., EMBO J 6 (1987) 43-48). The putative TATAA motif (CP Joshi, Nucl. Acids Res. 15: 6648-6653 (1987)) starts at 32, but, as not found in many other plant genes, the transcription start site (FIG. 1). ), No recognizable CAAAT motif is found 60 bp upstream. ZmC5c lacks a clearly recognizable AATAAA polyadenylation site signal. This is not unusual: 30% of plant genes lack a recognizable AATAAA motif (BD Mogen et al., Plant Cell
2 (1990) 1261-1272). In ZmC5c, a stretch of five A residues 16 bp upstream from the polyA addition site may act as a polyadenylation site, while other pollen-specific transcripts may have a position between the AATAAA motif and the poly (A) ⁺ addition site. (Tebbutt et al., Supra).

(Example 2) (Deduced amino acid sequence of ZmC5) The predicted amino acid sequence (563 amino acids) of ZmC5 was converted to EMBL and Ge.
Comparison with the nBank database revealed a high degree of homology to both plants (between 30.9% and 41.4%) and microbial PME (between 18.6% and 20.8%). Alignment of the amino acid sequences demonstrated conservation across both plant and microbial sequences that was primarily restricted to the C-terminus of the protein, which contains four regions that appear to be the catalytic domain or active site of the enzyme. (D. Alb
ani et al., Plant MoI Biol (1991) 16: 501-513, O.
Marcovic et al., Protein Sci 1: 1288-1292 (19
92)). A. niger PME in vitro mutagenesis (B Duwe et al., Bi).
othertechnol. Letts 18: 621-626 (1996) states that a histidine residue within region I, which is conserved in ZmC5, can be located at the active site of the enzyme and niger indicated that enzyme activity was required. However, this histidine has some PMEs of both plant and fungal origin.
Is replaced by another amino acid residue, which means that all PMEs
Suggests that it is not essential. In comparison to the plant PME, ZmC5 is than for the "early" pollen expressing Bp19 gene of D. napus (D. Albani et al., supra). inflata "late" pollen expressing PPE gene (J
H Mu et al., Plant MoI Biol 25: 539-544 (1994)).
Show a close relationship with

(Example 3) (Evaluation of the number of ZmC5 genes) 15 μm digested with any of BamHI, EcoRI, or HindIII
g of corn (inbred A188) genomic Southern blot containing
Probed with a radiolabeled full length ZmC5 cDNA insert. Two strong hybridizing bands in each lane of the blot in FIG. 2 indicate the presence of at least two similar genes in the corn genome.
Several additional bands showing much weaker signals suggest that this gene family may also contain some less related members.

Example 4 Spatial and Temporal Expression of ZmC5 A Northern blot containing 10 μg of total RNA from eight corn tissues was probed with cDNA ZmC5c to determine the gene expression program. A transcript of about 2.0 kb was detected only in pollen and budding pollen (FIG. 3 (A
)). This indicates that, within the limits of detection of this technology, the expression of this gene appears to be restricted to these two tissues. Leaves, roots, shoots, cobs
, Endosperm, or embryo, there was no detectable signal. The expression program during spikelet development was also determined. FIG. 3B shows 0.25, 0.5 and 1.0 cm.
1 shows a Northern blot containing total RNA from spikelets, mature pollen and budding pollen. The ethidium bromide stained gels demonstrate equal loading of RNA in each gel lane.

Spikelets were staged by staining the anthers with acto-carmine, and the anthers were found to contain cells at the following stages: premeiotic sporulation cells (pre) -Meitotic sporogenous cell (0.25 cm), early metaphase I (0.5 cm), mature pollen grains (1.0 cm). However, some overlap between successive stages is unavoidable due to variations in the developmental stages between two florets within the same spikelet. This Northern blot analysis shows that ZmC5 expression is restricted to mature cleaved and sprouting pollen without detectable expression in any other maize tissue, including spikelets containing cells in the early stages of microsporulation. It indicates that.

Example 5 Expression of ZmC5 Promoter / GUS Construct in Transgenic Plants Transcriptional fusions were made between the 5 ′ region of ZmC5g and the reporter gene β-glucuronidase (FIG. 4A), and the Agrobacterium trait Used for transformation of tobacco by transformation. This construct was made as follows: Two Sps
Using the hI site (one in the 2.5 kb SalI fragment containing the 2 kb sequence 5 ′ to the ATG and one in the polylinker), from position −61 to +403
The 3 ′ end of the position was removed (FIG. 1). This was combined with the SphI digested PCR fragment (regions -61 to +1 (an additional restriction site (BamHI, H
indIII, SalI), and a mutation to remove the HindIII site located at the transcription start site (FIGS. 1 and 4A)). The ZmC5 promoter region was then excised using the original 5 'SalI site and the introduced 3' SalI site and cloned into the Smal site of pGUS. The ZmC5 promoter-UID-A transcription fusion was then
The vector was transferred to the Bin19 vector (FIG. 4A) and used for Agrobacterium-mediated leaf-disk transformation of Nicotina tabacum var Samsun. Transformants were selected with kanamycin and the primary transgenic plants were regenerated. Two of them (positive for transgene expression) were sprouted to T2 generation.

[0063] Pollen grains from the cleaved anthers of the transgenic plants were collected and A.
Stained for GUS activity as described by Jefferson (Plant Mol Biol Rep (1987) 5: 387-405). Two plants were positive showing about 50% blue stained pollen (FIG. 4B). No blue coloration was detected in the non-transgenic controls. To investigate the number of integration sites, plants from the two transgenic lines were selfed and progeny were counted for resistance to kanamycin. Of the progeny evaluated from transgenic GC5-2, 303 plants were kanamycin resistant and 8 plants were kanamycin sensitive, giving an average ratio of 38: 1 indicating at least two integration sites. Progeny of the transgenic plant GC5-7 yielded an average ratio of kanamycin resistance to kanamycin sensitivity of 3.8: 1 indicating a single integration site (the expected ratio for one integration site was 3: 1, for two, 15:
63 for 1 and 3).

Extracts were made from a range of tissues containing five developmental stages of anthers and the fir G
US expression was analyzed fluorometrically (Jefferson, supra). FIG. 4 (C)
Shows GUS activity from two transgenic plants. Only very low levels of expression are detectable in tissues other than developing and mature cleaved tissues. In tobacco, the stage of bud development can be correlated with bud length (Teb
butt et al., supra), which depends on growth conditions. Thus, Bud-1 corresponds to microspores at the mitosis / tetrad stage, and Bud-2 corresponds to the mononuclear (unincleat).
e) corresponds to microspores, Bud-3 corresponds to microspore mitosis, Bud-4 corresponds to early to middle binuclear gametophyte, and Bud-5 corresponds to middle to late binuclear gametophyte. Corresponding. The microspore stage assessed by DAPI staining (data not shown) indicates that the time of expression of the ZmC5 promoter in tobacco is in good agreement with its expression in maize based on Northern data (FIG. 3; FIG. 4C). . Thus, in the native environment in both corn and transgenic tobacco, the ZmC5 promoter functions late in pollen development and is, in fact, inactive before microspore mitosis. Some variation in expression may be noted between the two transgenic lines with GC5-2, showing higher levels of expression in most tissues tested. Variations in expression levels are commonly found in transgenic populations and are based on the site of insertion of the transgene 9SLA (Hobbs et al., Plant Mol B
iol 21: 17-26 (1993)).

Example 6 Expression of GUS in Pollen Driven by an AlcA-Inducible Promoter A plant transformation vector containing the AlcA promoter driving GUS expression and the 35S CaMV promoter driving AlcR expression was obtained from tobacco. And has been introduced into tomato plants. GUS expression can be studied in all tissues before and after induction with ethanol as root drains.

GUS staining of tomato anthers and pollen shows clear expression of GUS after induction.
The same result is predicted from pollen from other species.

Example 7 Preparation of C5-Barnase Cassette (Predominant Gamete Male Sterility Cassette) The unique SalI site of pBluescript SK + (Stratagene) was replaced with the oligonucleotide linker MKLINK4 (5′-). TCGATTCG
Insertion of GCGGCCGCCGAA-3 ') into the digested SalI site
Replaced with NotI recognition site. A 0.9 kb BamHI-HindIII fragment carrying a barstar coding region driven by a bacterial promoter followed by a bacterial promoter was modified with a modified pBluescript.
Was inserted into the corresponding fragment. The nos terminator on the HindIII-NotI fragment was inserted into the corresponding fragment of the resulting vector. The undesired BamHI site is then inserted into the Str according to the manufacturer's instructions.
atagegene QuickChange system and oligonucleotide DAM-3A (5'-GGTCCGACTCTAGAGGAACCCCG
GGTACCAAGC-3 ′) and DAM-3S (5′-GCTTGGTAC)
CCGGGGTTCCTCTAGAGTCACCC-3 '). The resulting plasmid (designated pSK-BBN) was completely digested with BamHI and dephosphorylated with shrimp alkaline phosphatase (37 ° C., 1
time). A 1.9 kb BamHI fragment of the 5 ′ flanking region of C5 was ligated to it and subsequently digested with BamHI and PstI to confirm the presence and orientation of the insert, respectively. The resulting plasmid was named pSK-C5-BBN (FIG. 6). The entire cassette is then removed as an EcoRI-NotI fragment, resulting in the binary plant transformation vector pVB6. This construct is then introduced into Agrobacterium Tumefaciens by a freeze-thaw method. This DNA is introduced into tobacco using standard techniques.

Example 8 Analysis of Sterile Transgenic Plants Primary transformants are selected for growth on kanamycin in tissue culture, and this is confirmed by PCR analysis. The plants are grown and matured in a greenhouse. Pollen is collected from the anther after cleavage and live strains are used to determine whether the pollen is fertile or sterile. 50% of the pollen is expected to be sterile. Backcrossing of these plants to wild-type plants (after removal of anthers) or self-pollination results in progeny in which 50% of the pollen is sterile.

[0069] Other modifications to the invention that do not depart from the scope of the invention will be apparent to those skilled in the art.

[Brief description of the drawings]

FIG. 1 shows an alignment of ZmC5 cDNA with a 2.4 kb fragment from the 5 ′ region of its corresponding gene. The transcription start point is indicated ( ^* ) and the predicted translation start is underlined. Sa = SalI, S = SmaI, Sp = S
phI, H = HindIII, X = XhoI, P = PstI.

FIG. 2 shows a Southern blot of corn (inbred A188) genomic DNA. Each lane is EcoRI for lane 1 and Hin for lane 2.
Contains 15 μg of genomic DNA digested with dIII and for lane 3 with BamHI. Southern blots were hybridized with a radiolabeled ZmC5 cDNA probe.

FIG. 3 shows an ethidium bromide stained gel, which shows a Northern blot of the same gel probed with 18S RNA and ZmC5 cDNA probe of total RNA from various corn tissues. (A) Gels were loaded with 10 μg of total RNA from various corn tissues.
(B) The gel was filled with 10 μg of total RNA isolated from shoots, a series of spikelets during development, pollen, and budding pollen.

FIG. 4A is a physical map of the ZmC5 :: uidA construct and the junction sequence.
A residues indicated by an asterisk, an upward arrow and a black diamond, respectively, correspond to the ZmC5 transcription start site and are used to remove the HindIII site (for ease of cloning).
It corresponds to the T to A mutation and the 3 'end of the ZmC5 promoter region. ui
The dA translation start point is underlined.

FIG. 4B shows histochemical analysis of GUS activity in pollen from transgenic ZmC5 :: uidA tobacco, showing separation of blue staining.

FIG. 4C shows Zm in transgenic tobacco tissues from two transgenic lines (GC5-2 (open columns) and GC5-7 (black columns)).
4 shows the promoter activity of C5. Data from each line represents the average of two independent assays (normalized to non-transgenic controls). The stages of buds are as follows: Bud-1 corresponds to 5-8 mm buds (meiosis / tetraspore stage microspores), Bud-2: 10-12 mm buds (mononuclear microspores), Bud-
3: 13 to 15 mm (microspore mitosis), Bud-4: (17 to 22 mm) early to middle binuclear gametophyte, Bud-5: (27 to 45 mm) middle to late binuclear gametophyte (Tebutut) Et al., Supra).

FIG. 5 shows DNA encoding the ZmC5 promoter sequence in maize.
Shows the sequence. The underlined A is the putative transcription start point, and the bold and underlined ATG is the translation start point.

FIG. 6 shows an expression cassette containing C5-barnase / barstar-nos.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE , KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (71) Applicant Fernhurst, Haslemere, Surrey GU27 3JE, United Kingdom ( 72) Inventors Rogers, Hilary Johann, UK 13 Thiel Cardiff, P.K. Oh. Box 915, University of Wales, within the School of Pure and Applied Biology (72) Inventor Husey, Patrick Joseph, United Kingdom Tidable 200 F-term in Biological Sciences (Reference) 2B030 AA02 AB03 AD20 CA06 CA17 CA19 CB02 CD14 CG01 HA01 4B024 AA08 AA11 AA20 BA07 BA79 CA04 CA09 DA01 DA05 EA04 FA02 FA10 GA27 HA13 HA14 4B065 AA11 BAA20 ACA

Claims (27)

[Claims] 1. A recombinant nucleic acid sequence comprising the promoter sequence of the ZmC5 gene in maize, or a variant or fragment thereof that acts as a promoter in pollen. 2. The recombinant nucleic acid sequence of claim 1, comprising about 2 kb upstream of the transcription start site of the maize ZmC5 gene shown in FIG. 3. A recombinant nucleic acid sequence according to claim 1 or claim 2, wherein
It includes at least a part of the DNA sequence shown in FIG. 5, or a promoter sequence containing at least a part of a sequence encoding a promoter having substantially the same activity as the DNA sequence shown in FIG. 5, or a modified or fragment thereof. , Recombinant nucleic acid sequence. 4. An expression cassette comprising a nucleic acid sequence according to any one of claims 1 to 3, wherein said cassette is desired to be expressed in pollen and has an effect on pollen production. An expression cassette which is arranged to control a gene which may encode a product, an insecticidal toxin, or which enhance or modify the nutritional value of pollen. 5. The method of claim 4, wherein the gene comprises a gene that is harmful to pollen development.
An expression cassette according to item 1. 6. The expression cassette according to claim 5, wherein said gene is barnase, adenine nucleotide translocator, mutant tubulin, T-urf or trehalose phosphate phosphatase (TPP);
Or an expression cassette containing a gene encoding any of the ribozymes. 7. The expression cassette according to claim 4, wherein said gene comprises a selectable marker gene. 8. The expression cassette according to claim 7, wherein said selectable marker gene comprises an antibiotic resistance gene. 9. An expression cassette comprising the expression cassette according to claim 4.
Expression system. 10. An expression system according to claim 9, comprising a gene that is harmful to pollen and operably interconnected with a chemically inducible promoter, capable of overcoming the effects of said harmful gene. Or an expression system further comprising an expression cassette comprising a second nucleic acid sequence encoding a protein. 11. The expression system according to claim 10, wherein said nucleic acid sequence comprises a restoration gene. 12. The expression system according to claim 11, wherein the recovery gene is bulstar or TPS. 13. The expression system of claim 10, wherein the nucleic acid sequence encodes a construct that is sense or antisense to the deleterious gene, thereby suppressing its expression. 14. The expression system according to claim 10, wherein the second nucleic acid sequence is operably linked to the nucleic acid sequence according to any one of claims 1 to 3. Encoding a repressor protein for lac, tet, 434, lac-His or Amplicon ^™ that interacts with the sequence, thereby preventing expression of a first gene that is desired to be expressed in pollen;
Expression system. 15. The expression system according to claim 10, wherein the inducible promoter is an AlcA / R, GST, or ecdysone-inducible promoter. 16. The method according to claim 9, further comprising a selection marker.
An expression system according to the item. 17. The gene desired to be expressed in pollen,
The expression system according to any one of claims 9 to 16, which is linked to a herbicide resistance gene. 18. The following: (a) a first promoter sequence that is specifically expressed in pollen; (b) when expressed, disrupts pollen biosynthesis under the control of the pollen-specific promoter (C) a second promoter sequence responsive to the presence or absence of an exogenous chemical inducer; and (d) an element capable of inhibiting the expression of any of the first genes; Or a second gene operably linked to the second promoter sequence and encoding an element capable of inhibiting the first protein under the control thereof. 19. A method for producing a plant, said method comprising:
A method comprising transforming a plant cell using the expression system according to any one of the above. A plant cell comprising the expression system according to any one of claims 9 to 18. 21. A plant comprising the cell according to claim 20. 22. A method for inducing male sterility in a plant, the method comprising the step of transforming the plant with the expression system according to any one of claims 10 to 18. ,Method. 23. A method for controlling fertility of a plant, the method comprising:
19. A method comprising transforming the plant with the expression system of claim 1 or 18 and activating the inducible promoter if fertility is required to be restored. . 24. The method of claim 23, wherein said inducible promoter is activated by applying a chemical to said plant. 25. It comprises the recombinant nucleic acid according to any one of claims 1 to 3,
Replicable viral RNA vector (Amplicon ^™ ). 26. A method for transforming pollen, which comprises the step of transforming pollen cells using the expression system according to claim 9 or 18. 27. A recombinant nucleic acid, expression cassette, expression system, or method substantially as herein described with reference to any one of the accompanying drawings.